MDR1 has been described as an efficient, amplifiable, selectable marker in mammalian gene transfer experiments. Kane, S. E., et al., Gene 84:439-446 (1989). MDR1 based retroviruses have been used in mouse bone marrow transduction and reconstitution experiments to demonstrate long-term survival and in vivo selection of MDR1-marked hematopoietic cells. Sorrentino, B. P., et al., Science 257:99-103 (1992).
Existing MDR1 based plasmids, such as the plasmid HaMDR/A (available from NIH) are large and cumbersome. Pastan, I., et al., Proc. Natl.Acad. Sci.USA 85:4486-4490 (1988). The size of existing MDR1 plasmids may limit the known utility of MDR1 as an effective selectable marker in both in vitro and in vivo gene transfer experiments. Cloning manipulations involving such plasmids are difficult.
Current methods for using MDR1 to coexpress heterologous cDNAs in retroviral constructs require placing the heterologous cDNAs directly downstream of the MDR1 cDNA, for coexpression with MDR1 as a fusion gene and fusion protein. Germann, U. A., et al., FASEB J. 4:1501-1507 (1990). This construct may place severe limitations on the size of the fusion gene to be expressed. In addition, the eventual localization of the fusion gene product is limited, and independent regulation of expression of MDR1 and the heterologous gene of interest is not possible.
The amount of viral genetic information in known MDR1 based plasmids precludes insertion of a separate heterologous promoter and cDNA sequence between the retroviral long terminal repeats (LTRs) of the plasmid. However, placement of such sequences between the LTRs is a prerequisite to packaging into infectious retroviral particles for gene transfer to mammalian cells.
This invention reduces the overall size of the MDR1 plasmid and permits the insertion of a separate promoter to control expression of the heterologous cDNA.
The plasmids of the invention allow simultaneous expression and transfer of MDR1 (a selectable marker) plus other genes via retrovirus.